The project described in this proposal will study the processing of poliovirus proteins during a viral infection. A molecular genetic approach with infectious cDNA clones of the viral RNA as targets for site-specific mutagenesis will be used to define the specific regions of the viral genome involved in the enzyme-substrate interactions of proteolytic processing. A series of defined, mutant polio plasmids will be analyzed for phenotypic and biochemical expression in the following four different assay systems: (1) transfection of eukaryotic cells with polio plasmids, (2) rescue of proteinase mutants of infectious recombinant plasmids by co-transfection with simian virus 40 (SV40) vectors that express the P3 (replicase) region polypeptides of poliovirus, (3) bacterial expression of specific poliovirus polypeptides, and (4) in vitro translation of mutant mRNAs derived from bacteriophage SP6-specific transcription of polio cDNA. The proposed experiments will define the functional domains of the viral proteinase as well as those regions of polio proteins that are important in the formation of authentic substrate polypeptides. The data obtained should provide mechanisms for how the regulation of cleavage of poliovirus precursor polypeptides by a small genomic RNA can control both viral and cellular biosynthetic activity. Such a unique regulation of gene expression must contribute to the effectiveness of poliovirus and the other picornaviruses as cellular pathogens.